Chromosomes are constantly challenged by exogenous and endogenous threats. The repair of damaged DNAs is therefore crucial for maintaining genome stability. Improper DNA damage response induces genomic instability, resulting in cancer development. To maintain genomic integrity, all organisms respond to DNA damage by promptly launching the DNA-damage response. This response involves the recruitment of DNA repair factors to sites of DNA damage and the activation of signal transduction pathways, often termed DNA-damage checkpoint pathways. Checkpoint signaling requires two evolutionarily conserved phosphatidylinositol 3-kinase (Pl3K)-related protein kinases: ATM and ATR. While ATM responds primarily to DNA double-strand breaks, ATR recognizes various types of DNA lesions with single-stranded DNA. In the budding yeast Saccharomyces cerevisiae ATM and ATR correspond to Tel1 and Mec1, respectively. The long-term goal of this project is to uncover the regulatory mechanism of how Mec1 and Tel1 contribute to genome stability maintenance. In this proposal, we plan to uncover the molecular detail of how Mec1 activates the DNA damage checkpoint pathway (Aim 1), and define how Tel1 stimulates DNA damage signaling and regulates DNA break repair (Aim 2). We will also determine how Mec1 and Tel1 undergo protein maturation (Aim 3). Given the evolutionary conservation of DNA repair and checkpoint proteins, our study using budding yeast will provide invaluable information to understand how human ATM and ATR activates checkpoint signaling and controls DNA damage responses. Since improper DNA damage response and mutation accumulation are implicated in carcinogenesis and cell senescence, our study will contribute to the development of better cancer treatment and the prevention of premature aging.